vps35l Antibody

What is VPS35L and why is it important in cellular research?

VPS35L is a protein known as "VPS35 endosomal protein sorting factor like" in humans and may also be referred to as C16orf62, UPF0505 protein C16orf62, or esophageal cancer associated protein. Structurally, this protein has a molecular mass of approximately 109.6 kilodaltons . VPS35L functions as a crucial subunit of the Retriever complex, which plays an essential role in endosomal protein sorting. Research has identified VPS35L as the third responsible gene for Ritscher-Schinzel syndrome (RSS), following WASHC5 and CCDC22 . Understanding VPS35L is particularly important because disruption of its function can lead to various clinical manifestations, including hypercholesterolaemia, hypogammaglobulinaemia, and intestinal lymphangiectasia .

What species orthologs of VPS35L are available for cross-species research?

Based on gene homology, researchers can study VPS35L orthologs in various model organisms including canine, porcine, monkey, mouse, rat, and zebrafish systems . This cross-species availability is valuable for comparative studies and developing animal models of VPS35L-related disorders. When selecting antibodies for such research, it's important to verify species reactivity as suppliers offer VPS35L antibodies with different species specificities, including human-specific and zebrafish-specific variants .

What is the subcellular localization of VPS35L and how can it be visualized?

VPS35L primarily localizes to endosomes, as demonstrated by its colocalization with the early endosomal marker EEA1 . For visualization, immunocytochemistry (ICC) and immunofluorescence (IF) techniques using specific anti-VPS35L antibodies are effective approaches. These techniques typically involve cell fixation, permeabilization, blocking with appropriate buffers, overnight incubation with primary VPS35L antibodies, followed by fluorescently labeled secondary antibody incubation . For optimal results, researchers should use antibodies specifically validated for ICC/IF applications, as indicated in product specifications from suppliers like Novus Biologicals .

Which experimental techniques are most effective for studying VPS35L protein expression?

Multiple techniques are effective for studying VPS35L protein expression, with Western blotting (WB) being particularly valuable for quantification. When conducting Western blot analysis, proteins should be resolved on NuPAGE 4%–12% precast gels and transferred onto polyvinylidene fluoride membranes . After blocking with 5% skim milk in PBST, membranes should be incubated with primary VPS35L antibodies, followed by Alexa Fluor secondary antibodies for visualization using an infrared scanning system . For studying tissue distribution and expression patterns, immunohistochemistry (IHC) using paraffin-embedded sections (IHC-p) is recommended, with antibodies specifically validated for this application .

How can researchers assess VPS35L interactions with other Retriever complex components?

Immunoprecipitation assays are the gold standard for studying protein-protein interactions involving VPS35L. To assess interactions between VPS35L and other Retriever components like VPS29 and VPS26C, researchers should:

• Express VPS35L-GFP fusion proteins in appropriate cell lines

• Lyse cells in Tris-based immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 0.5% NP-40, with protease inhibitor cocktail)

• Incubate lysates with GFP trap beads for 1 hour at 4°C

• Wash beads three times with immunoprecipitation buffer

• Resolve proteins on SDS-PAGE gels and perform Western blotting

• Probe membranes with antibodies against interacting partners (e.g., VPS29, VPS26C)

This methodology has successfully demonstrated that VPS35L variants affecting the C-terminal region often disrupt binding to VPS29 while maintaining interaction with VPS26C .

What methods can be employed to study VPS35L function in lipoprotein receptor regulation?

To investigate VPS35L's role in regulating lipoprotein receptors, researchers can employ a combination of biotinylation assays and DiI-LDL uptake assays:

Biotinylation Assay Protocol:

• Culture VPS35L-WT, VPS35L-KO, or VPS35L-KD cells in DMEM with 10% lipid protein-deficient human serum for 12 hours

• Wash cells and incubate with Sulfo-NHS-SS-Biotin solution for 30 minutes

• Quench the reaction and collect cells by scraping in TBS

• Lyse cells and incubate clarified lysates with streptavidin beads

• Analyze both whole cell lysate and cell surface protein fractions by Western blotting for LDLR and LRP1

DiI-LDL Uptake Assay:

• Culture cells in DMEM with 10% lipid protein-deficient serum for 16 hours

• Incubate with DiI-LDL (5 μg/mL) in 5% LPDS/DMEM for 30 minutes

• Fix cells and mount with DAPI

• Quantify fluorescence intensity using ImageJ software, normalizing to the number of DAPI-stained nuclei

These complementary approaches have revealed that VPS35L ablation decreases cell surface levels of lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor (LDLR), resulting in reduced LDL cellular uptake .

How can researchers generate and express VPS35L variants to study structure-function relationships?

To study the functional consequences of VPS35L variants, researchers should:

• Clone human VPS35L cDNA into a lentiviral vector (e.g., pLVX-GFP-puro)

• Generate variant constructs using Gibson assembly

• Verify all constructs by DNA sequencing

• Produce lentivirus by co-transfecting the VPS35L expression vector with helper plasmids (Pax2 and pMD2G) into HEK293T cells using polyethylenimine

• Transduce target cell lines with the lentivirus

• Select stable cell lines using puromycin

This approach has been successfully used to express wild-type VPS35L and variants including N996del, M1020FS, A526_K550del, P876L, and A919T, enabling comparative functional analyses .

What methods are appropriate for analyzing mRNA expression and nonsense-mediated decay of VPS35L transcripts?

For comprehensive analysis of VPS35L mRNA expression and nonsense-mediated decay (NMD):

• Establish lymphoblastoid cell lines (LCLs) from patient samples

• Extract RNA and perform RT-PCR using VPS35L-specific primers

• Analyze expression patterns by Sanger sequencing of cDNA

• To detect transcripts subject to NMD, treat cells with cycloheximide (CHX) to prevent NMD

• Compare expression patterns between untreated and CHX-treated cells

• For detailed characterization of splice variants, perform TA cloning and sequence analysis of RT-PCR products

This methodology has revealed that certain VPS35L variants affect splicing and can generate alternative splicing products, such as a 75 bp in-frame deletion resulting in a protein with a 25 amino acid deletion (VPS35L-A526_K550del) .

How can researchers quantitatively compare VPS35L protein expression levels between different samples?

To accurately compare VPS35L protein expression levels:

• Prepare cell lysates under standardized conditions using appropriate lysis buffers

• Determine protein concentration using a reliable method (e.g., BCA assay)

• Load equal amounts of total protein for SDS-PAGE

• Transfer proteins to membranes and perform Western blotting with anti-VPS35L antibodies

• Include appropriate loading controls (e.g., β-actin, GAPDH)

• Visualize protein bands using an infrared scanning system

• Quantify band intensities using image analysis software

• Normalize VPS35L signal to loading control signals

This approach has demonstrated that VPS35L expression levels can significantly differ between patients with different clinical presentations, with more severe phenotypes correlating with lower VPS35L protein expression .

How should researchers address potential cross-reactivity issues when using VPS35L antibodies?

To address cross-reactivity issues:

• Validation controls: Include positive controls (cells known to express VPS35L), negative controls (VPS35L knockout cells), and isotype controls

• Antibody specificity testing: Test antibody specificity by immunoblotting lysates from cells overexpressing VPS35L and related proteins

• Signal verification: Verify signals using multiple antibodies targeting different epitopes of VPS35L

• Peptide competition: Perform peptide competition assays where the immunizing peptide is used to block specific antibody binding

• Genetic knockdown: Compare antibody signals in wild-type versus VPS35L knockdown cells

Remember that VPS35L may also be known by alternative names such as C16orf62 or UPF0505 protein C16orf62, so cross-reference antibody specifications carefully to ensure target specificity .

How can researchers interpret inconsistencies between protein and mRNA expression data for VPS35L?

When faced with inconsistencies between protein and mRNA expression:

• Consider post-transcriptional regulation: VPS35L protein levels can be affected by mechanisms independent of mRNA levels

• Evaluate protein stability: Variants affecting VPS35L binding to other Retriever components (particularly VPS29) can significantly reduce protein stability

• Analyze NMD effects: Some variants may escape NMD despite introducing frameshifts, particularly those near the last exon

• Assess alternative splicing: Some variants can generate alternative splicing products that maintain partial protein function

• Quantify relative contribution: Use cycloheximide treatment to distinguish between transcripts subject to NMD versus stable transcripts

Research has shown that VPS35L mRNA levels may not be significantly decreased in patient-derived cells carrying certain frameshift variants, yet protein levels can be substantially reduced, highlighting the importance of analyzing both mRNA and protein expression .

What factors should be considered when correlating VPS35L experimental findings with clinical phenotypes?

When correlating experimental findings with clinical phenotypes:

• Residual protein function: Even low levels of functional VPS35L protein may be sufficient to prevent severe manifestations

• Domain-specific effects: Variants affecting different domains may disrupt specific functions while preserving others

• Interaction profiles: Analyze how variants affect interactions with other Retriever components (VPS29, VPS26C)

• Cellular processes: Assess effects on specific cellular processes such as lipoprotein receptor trafficking

• Tissue-specific factors: Consider that VPS35L function may have different importance in different tissues

• Modifier genes: Acknowledge that genetic background may influence phenotypic expression

Research has demonstrated that patients with similar VPS35L variants can present with different clinical severities, and that residual VPS35L protein expression correlates with milder phenotypes in Ritscher-Schinzel syndrome .

What is the significance of VPS35L in Retriever complex function and endosomal sorting?

VPS35L serves as a critical scaffold protein in the Retriever complex, functioning alongside VPS26C and VPS29 to mediate endosomal protein sorting. Research findings indicate that:

• VPS35L localizes to early endosomes, colocalizing with EEA1

• The C-terminal region of VPS35L is particularly important for interaction with VPS29

• Mid-region domains mediate interaction with VPS26C

• Disruption of these interactions compromises Retriever complex stability and function

• Functional VPS35L is essential for proper trafficking of specific cargo proteins, including lipoprotein receptors

Studies of VPS35L variants have provided critical insights into structure-function relationships within the Retriever complex, demonstrating that the complex's assembly and stability depend on specific protein-protein interactions that can be disrupted by pathogenic variants .

How does VPS35L dysfunction contribute to the pathophysiology of Ritscher-Schinzel syndrome?

VPS35L dysfunction contributes to RSS pathophysiology through multiple mechanisms:

• Disrupted lipoprotein receptor trafficking: VPS35L ablation decreases cell surface levels of LRP1 and LDLR, resulting in reduced LDL cellular uptake and hypercholesterolaemia

• Impaired immune regulation: VPS35L-associated RSS exhibits hypogammaglobulinaemia not typically seen in RSS caused by WASHC5 and CCDC22 variants

• Vascular abnormalities: Intestinal lymphangiectasia appears to be a distinctive feature of VPS35L-associated RSS

• Renal involvement: Proteinuria has been observed in VPS35L-associated RSS but not in cases linked to other genetic causes

• Developmental impacts: The Retriever complex plays essential roles in multiple developmental processes

These findings establish VPS35L-associated RSS as a distinct clinical entity with a spectrum of phenotypic presentations ranging from severe to relatively mild, depending on residual protein function .

What are the most promising future research directions involving VPS35L antibodies?

Future research directions should focus on:

• Cargo identification: Using VPS35L antibodies for immunoprecipitation followed by mass spectrometry to identify novel cargo proteins regulated by the Retriever complex

• Tissue-specific functions: Investigating VPS35L expression and function across different tissues using immunohistochemistry

• Therapeutic potential: Exploring whether enhancing VPS35L stability or function could ameliorate disease manifestations

• Biomarker development: Evaluating whether VPS35L protein levels in accessible tissues correlate with disease severity

• Structural studies: Combining antibody-based approaches with structural biology techniques to elucidate the three-dimensional organization of the Retriever complex

• Pathway interactions: Investigating cross-talk between the Retriever complex and other endosomal sorting machineries

These research directions could significantly advance our understanding of endosomal sorting mechanisms and potentially lead to therapeutic interventions for VPS35L-associated disorders.